Experimental Induction of Bacterial Resistance to the Antimicrobial Peptide Tachyplesin I and Investigation of the Resistance Mechanisms

Abstract

Tachyplesin I is a 17-amino-acid cationic antimicrobial peptide (AMP) with a typical cyclic antiparallel ␤-sheet structure that is a promising therapeutic for infections, tumors, and viruses. To date, no bacterial resistance to tachyplesin I has been reported. To explore the safety of tachyplesin I as an antibacterial drug for wide clinical application, we experimentally induced bacterial resistance to tachyplesin I by using two selection procedures and studied the preliminary resistance mechanisms. Aeromonas hydrophila XS91-4-1, Pseudomonas aeruginosa CGMCC1.2620, and Escherichia coli ATCC 25922 and F41 showed resistance to tachyplesin I under long-term selection pressure with continuously increasing concentrations of tachyplesin I. In addition, P. aeruginosa and E. coli exhibited resistance to tachyplesin I under UV mutagenesis selection conditions. Cell growth and colony morphology were slightly different between control strains and strains with induced resistance. Cross-resistance to tachyplesin I and antimicrobial agents (cefoperazone and amikacin) or other AMPs (pexiganan, tachyplesin III, and polyphemusin I) was observed in some resistant mutants. Previous studies showed that extracellular protease-mediated degradation of AMPs induced bacterial resistance to AMPs. Our results indicated that the resistance mechanism of P. aeruginosa was not entirely dependent on extracellular proteolytic degradation of tachyplesin I; however, tachyplesin I could induce increased proteolytic activity in P. aeruginosa. Most importantly, our findings raise serious concerns about the long-term risks associated with the development and clinical use of tachyplesin I.A ntibiotic resistance in pathogenic bacteria and the emergence of superbacteria have attracted attention from health care workers worldwide. Antimicrobial peptides (AMPs) are promising candidates for development of novel alternative antibiotics. However, studies have indicated that some bacteria (especially human pathogens) are resistant to certain AMPs (1, 2), and bacterial resistance to cationic AMPs can arise through long and continual selection in the laboratory (3). Furthermore, some evidence demonstrates that bacteria can evolve resistance to AMPs after extended clinical application (4). Bacterial resistance to the AMPs nisin, pexiganan, and colistin has arisen through their clinical use. There is also evidence that pathogen resistance to AMPs may produce resistance to other AMPs with the same source and similar action mechanisms (5), or even to those with different sources and different action mechanisms (6, 7). Habets and Brockhurst previously reported that therapeutic use of pexiganan, representing a promising new class of AMPs, could drive the evolution of pathogens that are resistant to our own immunity peptides (6). Bacterial evolution of AMP resistance would greatly shorten and restrict the use of AMPs, so elucidation of the potential mechanisms of bacterial resistance to AMPs is required to inform the design of more effective drugs and to reduce the in...

Experimental Design, Population Dynamics, and Diversity in Microbial Experimental Evolution

“…Experimental evolution has also been carried out under more-challenging environmental conditions (Fig. 1D), for example, under extreme pH (100-103), osmotic pressure (101,(104)(105)(106), suboptimal redox status (101), oxygenation (107), extreme temperature (67,(108)(109)(110), or UV radiation (111); in the presence of antibiotics (50,(112)(113)(114)(115)(116)(117), antimicrobial peptides (118)(119)(120), and alcoholic solvents (101,121); and even under microgravity (122,123).…”

<p>Transcriptome Analysis Reveals the Resistance Mechanism of <em>Pseudomonas aeruginosa</em> to Tachyplesin I</p>

“…The PA-99 and PA-88 exhibited the increased levels of extracellular proteolytic activity and reduced the antimicrobial activity of tachyplesin I in previous studies. 14,15 Some DEGs were found to encode protease and peptidase in the present study, such as upregulated genes encoding protease (PA3913) and encoding peptidase (PA2658). The upregulated gene PA2315 and downregulated gene PA5514 both encoded beta-lactamase.…”

“…The minimal inhibitory concentration (MIC) values of tachyplesin I were 10 and 5 μg/mL for PA1.2620 and PA27853, respectively, in which these two strains were sensitive to tachyplesin I. Tachyplesin I-resistant mutants (PA-99 and P. aeruginosa ATCC27853-88-2 (PA-88)) were synthesized in our laboratory as described previously. 14,15 These strains were cultured in Mueller-Hinton broth (MHB) medium, and then seeded onto nutrient agar plates at 30°C or 37°C.…”

“…[11][12][13] Bacterial resistance to tachyplesin I has been induced in Aeromonas hydrophila XS91-4-1, P. aeruginosa CGMCC1.2620 (PA1.2620), P. aeruginosa ATCC27853 (PA27853), Escherichia coli ATCC25922 and F41 with crossresistance to other AMPs (e.g., pexiganan, tachyplesin III, and polyphemusin I), conventional antimicrobial agents (amikacin), as well as the potential involvement of extracellular proteases in mediating tachyplesin I-resistance in Gram-negative bacteria reported in previous studies. 14,15 A limited number of researches concentrated on the mechanism of bacterial resistance to tachyplesin I. [14][15][16] However, the resistance mechanism of P. aeruginosa to tachyplesin I has largely remained elusive.…”

“…14,15 A limited number of researches concentrated on the mechanism of bacterial resistance to tachyplesin I. [14][15][16] However, the resistance mechanism of P. aeruginosa to tachyplesin I has largely remained elusive.…”